We are applying proteomic methodology to unresolved problems in neuropathologic diseases. Progress continues in studies on the structure of the postsynaptic density and its remodeling by drugs used in the treatment of mood disorders, on protein complexes implicated by genomic studies of schizophrenia, and synaptic protein changes accompanying long term depression (LTD). The ErbB4 and Dystrobrevin binding protein 1 (dysbindin) protein signaling complexes have both been implicated by genomic studies on schizophrenia and have remained difficult to analyze in brain. We are continuing a project to identify, characterize and validate complexes associated with both proteins. ErbB4 is a receptor tyrosine-protein kinase, and we have used antibodies to covalently link it to magnetic beads. Comparative LC/MS/MS proteomic studies using on-bead digestion procedures developed for this project have failed to provide the enrichment factor required for ErbB4 recovery and mass spectrometric detection. However, new peptide targeting strategies are planned using labeled proteins from synthetic genes to allow better tracking of enrichment and recovery of this complex. Protein degradation/modification by proteases involves many physiological processes such as apoptosis. Caspase-3 is a cysteine protease best known as an executioner protease in apoptosis. Recent studies suggest that caspase-3 plays an important non-apoptotic function in synapses as well. In hippocampal neurons, caspase-3 activity is specifically required for NMDA receptor-dependent long-term depression (LTD), but not for long-term potentiation. More importantly, activation of casapse-3 in LTD promotes AMPA receptor endocytosis instead of cell death. To understand the molecular mechanism by which caspase-3 activation leads to LTD, we applied a published method developed to selectively capture emergent peptides derived from proteolytic cleavage upon NMDA receptor activation and apoptosis induction. Briefly, the newly formed free, unblocked protein N-termini produced from protease cleavage events are tagged with a biotinylated peptide using an engineered enzyme subtiligase. The labeled proteins are enriched by immobilized avidin and analyzed by mass spectrometry. The substrate degradomes of control (untreated neurons), NMDA treated, and staurosporine treated samples are compared. Currently, we are focusing on the substrates with putative caspase-cleavage sites and testing their functions in LTD.